Antibodies represent a large proportion of therapeutic drugs currently in development. Antibodies are complex glycoproteins that recognize and bind to target antigens with great specificity. This specific binding activity allows antibodies to be used for a range of applications, including the diagnosis, prevention and treatment of disease (20). Typical full size antibodies are tetramers of two identical heavy chains and two identical light chains. Beyond full size immunoglobulins, other antibody derivatives of therapeutic value have been expressed in plants, including Fab fragments, scFvs, bispecific Fvs, diabodies, minibodies, single variable domains, antibody fusion proteins and more (4).
Glycosylation of IgG antibody molecules is a post translational modification that is important for recognition of the effector ligands FcR and complement. A complex biantennary oligosaccharide moiety is attached at Asn-297 in the CH2 domain of each heavy chain. Heterogeneity in the attachment of sugar residues is associated with functional modulation (21-26). Furthermore, studies show that the in-vivo fate of IgG1 antibodies is drastically affected by the presence of a carbohydrate of altered structure in CH2 (27).
Proteins for pharmaceutical use have been traditionally produced in mammalian or bacterial expression systems. In most cases, they are produced transgenically in mammalian cell lines, primarily Chinese hamster ovary (CHO), or transgenic animals because these have been shown to fold and assemble the proteins correctly and generate similar glycosylation patterns. However, such expression systems are expensive and are difficult to scale up to high levels of production. Furthermore, there are safety concerns due to potential contamination with pathogenic organisms or oncogenic DNA sequences. Also, the production yield and stability of certain subclasses, for example IgG4, in these mammalian systems is quite low, such that production is very inefficient.
Although the exact cause is not known, the stability of the recombinant IgG4 is low, which causes the yield to be low, and thereby leads to inefficient large scale production.
Therefore, clearly non-mammalian systems for production of antibodies and other therapeutic proteins would be advantageous. Although such systems have been shown to be operative for non-immunogenic proteins, antibodies are more sensitive and more difficult to produce in non-mammalian cell culture systems. For example although antibodies can be expressed in baculovirus expression systems and stably transfected insect cell lines, the resultant material may not have the necessary properties. Insect cell expression systems do produce antibodies, but have several deficiencies: inefficient processing and an impairment of the folding and secretion capacity, a high, in part baculovirus-encoded, protease activity, insufficient strength and deviations of the posttranslational modification pattern (which could act immunogenically)(see for example the following references: Guttieri M C, Liang M. 2004, Human antibody production using insect-cell expression systems. Methods Mol. Biol., 248:269-99, Guttieri M C, Sinha T, Bookwalter C, Liang M, Schmaljohn C S. 2003, Cassette vectors for conversion of Fab fragments into full-length human IgG1 monoclonal antibodies by expression in stably transformed insect cells. Hybrid Hybridomics. 22(3):135-45, Potter K N, Li Y, Capra J D. 1993, Antibody production in the baculovirus expression system. Int Rev Immunol. 10(2-3):103-12). Antibodies cannot be produced in E. coli as there is no suitable post-translational modification.
In the past decade a new expression system has been developed in plants. This methodology utilizes Agrobacterium, a bacteria capable of inserting single stranded DNA molecules (T-DNA) into the plant genome (1). Due to the relative simplicity of introducing genes for mass production of proteins and peptides, this methodology is becoming increasingly popular as an alternative protein expression system in plants (2-4) (5).
Plant based systems represent an inexpensive, efficient and safe alternative for the production of recombinant antibodies. Production of full size antibodies in plant cells was first demonstrated in whole tobacco plants by sexual crossing of plants expressing single gamma or kappa immunoglobulin chains (6). Assembly of IgG (primarily IgG1) and IgA antibodies in Nicotiana, Arabidopsis and other plants has been described (3, 7-10).
Research over the last 10 years has shown that plant cells, contained in whole plants, can produce a variety of functional antibodies and there is now intense interest in scaling up production to commercial levels. (11-13), (14,15), (5, 16-18).
However there is rising concern about potential safety issues, including contamination with residual pesticides, herbicides and toxic plant metabolites, when using transgenic field crops to produce recombinant proteins (19). Groups opposed to genetically modified plants in general, afraid of the potential danger that transgenes and their encoded proteins will spread in the environment or into the food chain, and strict limitations of regulatory bodies have raised obstacles for companies utilizing transgenic plant technology for protein expression. Thus, clearly the use of whole, complete plants is disadvantageous.
Plant-suspension cells are an in vitro system that can be used for recombinant protein production under carefully controlled certified conditions. Plant cell suspensions can be grown in shaken flasks or bioreactors to produce recombinant proteins. The present inventors have filed corresponding applications for a bioreactor system which allows safe production of recombinant proteins, such as antibodies, utilizing the advantages of plant cell expression, without the potential hazard of open-field plant growth (see U.S. Pat. No. 6,391,638 and U.S. patent application Ser. No. 10/784,295, filed on Feb. 24, 2004, both of which are hereby incorporated by reference as if fully set forth herein).
For example, expression of a TMV-specific full-size murine IgG-2b/K antibody in a Nicotiana tabacum cv. Petite Havana SR1 suspension culture (P9s) has been described (18). The integration of an N-terminal murine leader peptide directed the assembled immunoglobulin for secretion. However, in suspension culture, the full-size recombinant antibody was retained by the plant cell wall. An ELISA procedure demonstrated that the specificity and affinity of the recombinant antibody was indistinguishable from its murine counterpart, indicating the potential of plant cell suspension cultures as bio-reactors for the production of recombinant antibodies (18).
The production of antibodies in plants represents a special challenge because the molecules must fold and assemble correctly to recognize their cognate antigens. On the other hand, plant derived expression systems do facilitate post-translational modifications known to be crucial for protein expression and activity, unlike bacterial expression systems for example. However, there are significant differences in post-translational modifications between mammalian and plant cell culture systems, which need to be considered in order to avoid potential reduced or even eliminated functionality of the expressed protein.
One of the major differences between mammalian and plant protein expression system is the variation of protein sugar side chains, caused by the differences in biosynthetic pathways. Glycosylation was shown to have a profound effect on activity, folding, stability, solubility, susceptibility to proteases, blood clearance rate and antigenic potential of proteins. Hence, any protein production in plants should take into consideration the potential ramifications of plant glycosylation.
Protein glycosylation is divided into two categories: N-linked and O-linked modifications (28,29) (30). The two types differ in amino acid to which the glycan moiety is attached to—N-linked are attached to Asn residues, while O-linked are attached to Ser or Thr residues. In addition, the glycan sequence of each type bears unique distinguishing features. Of the two types, N-linked glycosylation is the more abundant, and its effect on protein function has been extensively studied. O-linked glycans, on the other hand are relatively scarce, and less information is available regarding their affect on proteins.
Several approaches have been discussed to control and tailor protein glycosylation in plants (31) (32). Gross modifications, such as complete inhibition of glycosylation or the removal of glycosylation sites from the peptide chain, may be implemented as one strategy. However, this approach can result in structural defects. An additional approach involves knock-out and introduction of specific carbohydrate processing enzymes. These enzymes are “knocked-out” to prevent potentially immunogenic sugars from being added during post-translational modification. For example, knock-out of the gene encoding for Xylosyltransferase would result in the absence of xylose in the glycan structure. Xylose is a sugar residue found only in plants and is thought to be potentially immunogenic. Introduction of human carbohydrate processing enzyme genes such as sialyltransferase to the plant results in the addition of sialic acid, which is not present in plants (see for example Ragon C, Lerouge P, Faye L., 1998 The protein N-glycosylation in plants, J. Exp. Botany Vol 49(326)1463-1472).
The third approach tries to localize the expression to a specific compartment in the cell. For example, retaining the protein in the ER prevents plant specific modification from being carried out in the Golgi (33) (34,35). Each cellular compartment has different carbohydrate processing enzymes. Proteins that enter or are targeted to the secretory pathway are transferred from the ER to the Golgi and then to the vacuola or apoplast. The apoplast is the space between the plant cell membrane and plant cell wall. Proteins that are targeted to secretion, or more specifically, are not targeted to a specific cell compartment and are therefore secreted, reach the apoplast. Some proteins remain there but some are passed through the cell wall and are secreted to the growth medium. Since different carbohydrate processing occurs in each compartment, retaining a protein in one compartment can inhibit further processing of the glycan structure, or, by directing a protein to a specific compartment, it is possible to ensure that the protein enters a desired processing pathway.